This invention relates to the use of a very wide class of 2',5'-oligoadenylates and their analogs (2',5'-oligonucleotides) linked in a 2',5'-phosphodiester bond to inhibit viral diseases in plants, plant tissues, leaves and other parts.
In Devash et al, Science 216, 1415-1416 (1982), the disclosure of which is completely incorporated herein by reference, it was shown that 2',5'-oligoadenylates, a family of compounds which are induced in interferon treated animal cells, and their analogs (2',5'-oligonucleotides) is induced in interferon treated animal cells, protects plant tissue from infection by the tobacco mosaic virus (TMV). The basis 2',5'-oligoadenylate is referred to as 2,5-A or 2',5'A. This inhibition of virus multiplication was obtained in concentrations comparable to those affecting protein synthesis and antiviral activities in animal cells. After one hour treatment with (2',5') oligoadenylates, the multiplicity of tobacco mosaic virus was reduced by 80 to 90% with concentrations of 100 to 200 nM 2,5-A being sufficient to achieve near-total inhibition. However, it was noted that the 2,5A must be applied to the TMV infected tissue early in infection to obtain a maximum antiviral affect.
By virtue of the present invention the present inventor who is the lead author in the 1982 Devash et al. publication, has now determined there is a very wide class of 2',5'-oligoadenylates and 2',5'-oligonucleotide analogs linked in a 2',5'-phosphodiester bond which are effective in extremely low concentration to inhibit replication of tobacco mosaic virus (TMV) and a broad spectrum of viral diseases in plants.
It is known that protection is conferred to virus-infected mammalian cells by the formation of interferon which induces the synthesis of at lease two enzymes. One of these enzymes is the 2',5'-adenylate synthetase which in the presence of certain double-stranded RNAs converts adenosine triphosphate (ATP) to a series of unique 2',5'-oligoadenylates.
In addition to the synthesis of the 5'-triphosphate 2',5'-linked oligoadenylates, there have been reports concerning the intracellular accumulation of the 5'-dephosphorylated 2',5'-adenylate molecules (referred to here as 2',5'-adenylate cores) in mouse L-cells following treatment with interferon. It was concluded that because the amount of cores seemed to be independent of the concentration of the 2',5'-oligoadenylate 5'-triphosphate molecule, 2',5'-oligoadenylate cores may play a separate role in the inhibition of DNA-synthesis and cellular reactions.
Because of the rapid hydrolysis of the naturally occurring 2',5'-oligoadenylate molecule in cells, there have been reports on the enzymatic and chemical synthesis of the 2',5'-oligoadenylate, 5'-triphosphate analogs and their corresponding cores in attempts to increase the metabolic stability and retain inhibition of protein syntheses. In addition, it has been reported that the 5'-diphosphate of 3',5'-adenylate molecule activated the 2',5'-A.sub.n -dependent endonuclease to hydrolyze mouse L-cell rRNA. Also, the 2',5'-cordycepin tetramer 5'-triphosphate analog complexes with and activated the 2',5'-A.sub.n -dependent endonuclease to hydrolyze vesicular stomatitis virus mRNA and inhibit protein synthesis.
Because of the natural occurrence of the 2',5'-oligoadenylate core in mammalian cells, various laboratories have studied the effect of core compounds on cellular processes in animal cells, tumor growth in whole animals, and inhibition of virus replication in plants. For example, it has been demonstrated that the 2',5'-oligoadenylate cores and 2',5'-cordycepin trimer core inhibit transformation of Epstein-Barr virus-infected lymphocytes by inhibiting the synthesis of the Epstein-Barr virus-induced nuclear antigen, augment natural killer cell activity, inhibit TMV replication in tobacco plants, and inhibit chondrosarcoma growth in animals.
It has been demonstrated that the 2',5'-oligoadenylate core analogs do not exert their action by 5'-rephosphorylation followed by activation of the 2',5'-A.sub.n -dependent endonuclease. There have been suggestions that cores may act by pathways independent of interferon.
It has been further reported that 2',5'-cores act as prodrugs due to degradation by esterases. However, this does not appear to be the case in all mammalian cell systems. For example, it has been demonstrated that tritium-labeled 2',5'-cordycepin trimer core is taken up intact by human lymphocytes.
Recently, it has been reported that human recombinant leukocyte interferon inhibited TMV replication in tobacco leaf discs. Furthermore, the antiviral factor isolated from TMV-infected leaves of Nicotiana glutinosa and human leukocyte interferon induced double-standed RNA-dependent synthesis of oligoadenylates from ATP in plants, producing oligonucleotides with antiviral activity.
In the present invention a very wide class of oligodenylates and oligonucleotides analogs linked in a 2',5'-phosphodiester bond with a chain length of dimer up to ten nucleosides long, with or without external phosphates inhibit TMV replication in TMV-infected tobacco leaf discs, TMV-infected protoplasts, and in whole plants.
The adenosine molecule has the following structure: ##STR1## R' above is known as the aglycone moiety. R" above is known as the ribosyl or the ribosyl sugar moiety. It is well known that either the aglycone or the ribosyl can be modified in various ways. For instance, the 6-amino group on the aglycone (adenosine) can be substituted by a hydroxyl group to form inosine. Alternatively the 7 position nitrogen atom of adenosine can be substituted by a carbon atom to form tubercidin.
In the ribosyl, the hydroxyl group in the 3' position can be substituted by hydrogen to form cordycepin, or the hydrogen and the hydroxyl groups can change positions on the 2' carbon to form ara-A or a hydrogen and hydroxyl groups can change position on the 3' to form xylo-A.
Moreover, the inhibition effects observed in the present invention on plant viruses is applicable to all the foregoing variations in 2,5-A as well as other modifications of the basic adenosine structure regardless of the modifications on either the aglycone or the rybosyl or both so long as the oligoadenylate or the oligonucleotide analogs are linked in a 2',5'-phosphodiester bond.
The present invention also encompasses the so-called core compounds as well as the unique series of oligoadenylate 5'-triphosphate PPPA(2'p5'A).sub.n which are commonly known as 2,5-A. The core differs from 2,5-A itself or 2,5-A related compounds, since the core lacks the 5'-terminal triphosphates. The present invention also encompasses (1) the existence of any external phosphate (i.e., mono, di, tri and tetra phosphates present in either the 5',2' or 3' positions) (2) variations in the aglycone or ribosyl as discussed hereinabove. The present invention further includes the dimer, trimer, tetramer and up to 2',5'-oligonucleotides with a chain length of up to ten nucleosides, (r=10). The trimer is illustrated below: ##STR2##
In view of the foregoing it is very clear that the present invention applies to any oligomer of 2',5' of any size where r=2 to r=10.
Moreover, the present invention includes any modifications on the internal and external phosphodiester bonds, such as modifications on the bridged oxygens as well as any modifications on the non-bridged oxygen (double bond phosphorous). The present invention is also applicable to any change on the phosphonyl group.
Accordingly, by way of example only and without limitation it follows that there are many compounds covered by this invention, so long as there is the 2',5'-phophodiester bond as discussed herein above. Exemplorary of compounds falling within the scope of the present invention are 2',5'-adenylate dimer or trimer cores, 2',5'-cordycepin trimer cores, 2',5'-inosinate dimer trimer cores, adenylyl-(2' 5')-adenylyl-(2' 5')-9-.beta.-D-arabinofuranosyladenine; adenylyl-(2' 5')-adenylyl-(2' 5)-tubercidin.